Tg(mpeg1:dendra2) zebrafish embryos at 2.5 dpf were anesthetized using 0.16 mg/ml tricaine and then mounted in a confocal dish using 1% low-melting agarose. A Leica SPE confocal microscope was applied to induce photoconversion in the tail region, largely the caudal hematopoietic tissue (CHT), of embryos by using prolonged (approximately 1 min) illumination with a scanned 405-nm laser. For confirmation, the GFP and RFP signals were assessed in both the tail and head regions of the embryos using a Leica SPE confocal microscope after photoconversion.
Spe confocal microscope
Manufactured by Leica
Sourced in Germany, United States, France, Portugal
The Leica SPE confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a state-of-the-art confocal optical system that enables high-resolution, three-dimensional imaging of samples. The SPE confocal microscope is capable of producing detailed, high-quality images by precisely controlling the illumination and detection of light within the sample.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (4 mentions)
Vectashield, by Vector Laboratories (4 mentions)
Sp8 confocal microscope, by Leica (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Sp5 confocal microscope, by Leica (3 mentions)
Donkey anti rabbit alexa 594, by Thermo Fisher Scientific (3 mentions)
Fish skin gelatin, by Merck Group (3 mentions)
Prolong antifade medium dapi, by Thermo Fisher Scientific (3 mentions)
Live dead staining, by Thermo Fisher Scientific (3 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Spe microscope, by Leica (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Biotin wfa, by Merck Group (2 mentions)
Gephyrin, by Synaptic Systems (2 mentions)
198 protocols using spe confocal microscope
1
Photoconversion of Zebrafish Caudal Hematopoietic Tissue
2
Immunofluorescence Staining of Organoids
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Organoids were collected with cold PBS and transferred to a 15-ml tube. They were spun down at 300 g for 3’ and fixed with 4% PFA for 1 h at room temperature. After washing twice with PBS, organoids were permeabilized with PBS-1% Triton-X-100 for 20’ at 4 °C and incubated in blocking solution (PBS + 1% BSA + 3% goat serum + 0.2% Triton-X-100) for 1 h at 4 °C. Organoids were then incubated with primary antibodies in working solution (PBS + 0.1% BSA + 0.3% goat serum + 0.2% Triton-X-100) over night at 4 °C. The primary antibodies were removed, and the samples were washed three times for 5 min each with PBS before incubating them with secondary antibodies (1:500) in working solution for 1 h at room temperature. Organoids were stained with DAPI, resuspended with mounting medium and mounted on slides. Images were acquired using a Leica SPE confocal microscope, a Leica SP8 confocal microscope or an in house-built Olympus two-photon microscope (Multiphoton Microcopy Core, Massachusetts General Hospital), depending on the experiment. For live imaging of Pdk1-mCherry, organoids were plated in Matrigel drops in 6-cm plates and imaged by two-photon microscopy using a water immersion 25× objective. To measure ROS levels in organoids, we incubated them with CellRox Green (Life Technologies, C10444) for 1 h, fixed them with 2% PFA and imaged them using a Leica SPE confocal microscope.
3
Microglial Cell Transfection and Imaging
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Microglial cells were transfected in 24-well glass-bottomed plates (Greiner Bio-One, Ref. 662892) with a DNA mixture containing eGFP-DCAF1, mRFP-CTIP2 or HA-Vpr. At 24 h post-transfection, cells were extensively washed, fixed with 4% PFA/PBS solution and kept in 1x PBS at 4 °C until observation with a Leica SPE confocal microscope equipped with a Leica 63x1.4NA oil immersion objective (HXC PL APO 63x/1.40 OIL CS).
4
Immunofluorescence Imaging of Neuronal Synaptic Markers
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Neuronal cultures were fixed with 4 % paraformaldehyde and 4 % sucrose. The following antibodies were used: rabbit anti-CB1 (1:500; Synaptic System, Goettingen, Germany), guinea pig anti-vGLUT1 (1:1000; Synaptic System), and guinea pig anti -VGAT (1:750; Synaptic System). Secondary antibodies were conjugated with Alexa-488 and Alexa-555 fluorophores (Invitrogen).
Images were acquired by using a Leica Spe confocal microscope equipped with an ACS APO 63.0X1.3 objective (Leica, Wetzlar, Germany).
Pixel size was 94.8 nm_94.8 nm, and acquisition parameters (i.e., laser power, gain, and offset) were kept constant across different experimental settings. The minimum puncta size was set at three pixels.
Colocalization of two selected markers was measured by using the boolean function “and” for the selected channels. The resulting image was binarized, inverted, and used as a colocalization mask to be subtracted from single channel. The number of puncta resulting from colocalization mask subtraction (colocalizing puncta) was measured for each marker. A colocalization ratio was set as colocalized area/total puncta aerea. Fluorescence image processing and analyses were performed with the ImageJ Software (National Institutes of Health, Bethesda, MD, USA).
Images were acquired by using a Leica Spe confocal microscope equipped with an ACS APO 63.0X1.3 objective (Leica, Wetzlar, Germany).
Pixel size was 94.8 nm_94.8 nm, and acquisition parameters (i.e., laser power, gain, and offset) were kept constant across different experimental settings. The minimum puncta size was set at three pixels.
Colocalization of two selected markers was measured by using the boolean function “and” for the selected channels. The resulting image was binarized, inverted, and used as a colocalization mask to be subtracted from single channel. The number of puncta resulting from colocalization mask subtraction (colocalizing puncta) was measured for each marker. A colocalization ratio was set as colocalized area/total puncta aerea. Fluorescence image processing and analyses were performed with the ImageJ Software (National Institutes of Health, Bethesda, MD, USA).
5
Visualizing Receptor Populations in Cells
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These assays were done exactly as previously (Ghosh et al., 2010 (link); Garcia-Marcos et al., 2011a (link)), using a confocal Leica SPE microscope. To visualize all populations of receptors in cells, images were acquired as Z-stacks and maximally projected. Briefly, cells were fixed at room temperature with 3% paraformaldehyde for 20–25 min, permeabilized (0.2% Triton X-100) for 45 min, and incubated for 1 h each with primary and then secondary antibodies as described previously (Ghosh et al., 2008 (link)). Antibody dilutions were as follows: mAb green fluorescent protein, 1:500; secondary goat anti-rabbit (594) and goat anti-mouse (488) Alexa-conjugated antibodies, 1:500; and 4′,6-diamidino-2-phenylindole (DAPI), 1:2000 (Molecular Probes). Samples were examined with a Leica SPE confocal microscope (Leica) using a 63× objective, and collected images were processed using ImageJ software (National Institutes of Health, Bethesda, MD) and assembled for presentation using Photoshop and Illustrator software (Adobe).
6
Immunofluorescence Staining Protocol for Hippocampal Neurons
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Immunofluorescence staining was carried out as described in (Verderio et al., 1994 (link)) using the following antibodies: PSD-95 (1:400; monoclonal; UC Davis/NIH NeuroMab Facility, CA), MeCP2 (1:200; polyclonal; Cell Signaling), MAP2 (1:300; monoclonal; Immunological Sciences). Images were acquired using a Leica SPE confocal microscope. Images of primary hippocampal cultures were acquired with a Leica SPE confocal X 63 oil immersion lens (1,024 × 1,024 pixels). Each image consisted of a stack of images taken through the z-plane of the cell. Confocal microscope settings were kept the same for all scans in each experiment.
7
Immunofluorescence Analysis of Synaptic Puncta
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Sections were blocked with 5 % normal goat serum (NGS) or normal horse serum (NHS) in PBS-T for 1 h at RT. The primary antibodies (PV; 1:1000, Swant CS56; 1:100, Sigma-Aldrich; Biotin-WFA; 1:100, Sigma-Aldrich, Gephyrin; 1:200, Synaptic system) were incubated overnight at 4 °C. Following three washes in PBS they were incubated for 2 h at RT with the appropriate secondary antibody conjugated with Alexa fluor 647, Alexa fluor 488 or Alexa fluor 568 or Streptavidin-Alexa fluor 647 (Molecular Probes, Invitrogen) diluted 1:500 in PBS-T. incubated with secondary antibodies for 2 h. Sections were rinsed and mounted on 1% gelatin-coated slides with FluorSave™ Reagent (Merck Millipore, Germany).
For synaptic puncta, quantification images were captured using a Leica SPE confocal microscope using ×63 objectives with a 1024 × 1024 image resolution (n = 6 per group). At least 3 z-stack images (total 5 µm) were taken per section with at least three sections analyzed per animal (~360 µm apart). Images contained at least 5 PV positive neurons. At least 50 PV+ neurons per animal were analysed for Gephyrin (+) puncta quantification. Synaptic puncta analysis was performed with an automated custom script using an imageJ 1.29 plugin (available from c.eroglu@cellbio.duke.edu) [64 ].
For synaptic puncta, quantification images were captured using a Leica SPE confocal microscope using ×63 objectives with a 1024 × 1024 image resolution (n = 6 per group). At least 3 z-stack images (total 5 µm) were taken per section with at least three sections analyzed per animal (~360 µm apart). Images contained at least 5 PV positive neurons. At least 50 PV+ neurons per animal were analysed for Gephyrin (+) puncta quantification. Synaptic puncta analysis was performed with an automated custom script using an imageJ 1.29 plugin (available from c.eroglu@cellbio.duke.edu) [64 ].
8
Transcardial Perfusion and Brain Imaging
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Animals were anesthetized (25% ketamine and 5% xylazine solubilized in a NaCl solution) and perfused with 25 ml cooled phosphate buffer saline (PBS) and 50 ml 4% PFA. Then, the brains were treated as previously described by Heise et al. [24 (link)]. Images of immunostained sections were taken using an upright fluorescence microscope (Axioskop, Zeiss) and Axiovision software (Zeiss). For in vivo studies, a confocal microscope (Leica SP5 or Leica SPE confocal microscope with a ×40 or ×63 objective) was used. For the magnified images, Fiji ImageJ (National Institute of Health, USA) and Bitplane Imaris software were used.
9
Retinal Angiogenesis Analysis Protocol
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To analyse retinal angiogenesis, the procedures of isolation and staining of the retinas were performed as published (Pitulescu et al., 2010 (link)). Briefly, retinas were dissected in PBS and blocked/permeabilized in retina blocking buffer (1% BSA and 0.3% Triton X-100 in PBS) for 1–2 hr at room temperature. Alexa Fluor 488 conjugated Isolectin GS-IB4 (Invitrogen) diluted in Pblec solution (1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2 and 1% Triton X-100 in PBS) was added to visualize whole-retina vasculature by incubating overnight at 4°C, followed by staining for ESM1 (primary goat anti-ESM1 antibody; R and D Systems). After mounting, images of retinas were taken using a Leica SPE confocal microscope equipped with a HC PL APO 20X/0.75 IMM CORR CS2 objective or Leica SP8 confocal microscope equipped with a HCX IRAPO L 25X/0.95 W objective. Images were taken at room temperature using Leica LAS X software and processed with image J software.
10
Schwann Cell Migration in Nerve Regeneration
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Rat pups were obtained from the Plymouth Medical School Animal Facility and euthanized by cervical dislocation. Rat Schwann cells were prepared from the sciatic nerve and brachial plexus of 3-day-old Sprague-Dawley rat pups. Mouse Schwann cells were cultured from the distal sciatic nerve 7 days after transection injury (Woodley et al., 2019). Tibial nerves from GFP-positive control or Runx2 KO mice were cut into 2-mm pieces in a petri dish with a surgical blade. Nerve explants were transferred to 24-well plates and cultured in 1 mL low-glucose (1 g/mL) DMEM (D2902, Sigma) containing 10% FBS for 150 hours. GFP-positive migrating Schwann cells from tibial nerve explants were imaged using a LeicaIM8 fluorescence microscope (Leica, Wetzlar, Germany). Migrating Schwann cells were counted after the images were taken. In vivo Schwann cell migration was assessed at day 6 following sciatic nerve transection injury in both GFP-positive control and Runx2 KO mice. On day 6, transected sciatic nerves were harvested and fixed in 4% paraformaldehyde (in PBS, PH7.2, Sigma) at 4°C overnight. Nerves were cleared with 25%, 50%, and 75% glycerol (# 56-81-5, Sigma, in PBS) for imaging with a LeicaSPE confocal microscope (Leica). Z-stack images were taken and combined to measure the area of migrating Schwann cells (GFP-positive) in the nerve bridge.
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